The relationship between structure and function of molecules is a fundamental issue in the study of biological systems. Structure-function relationships are important in understanding, for example, the function of enzymes, cellular communication, and cellular control and feedback mechanisms. Certain macromolecules are known to interact and bind to other molecules having a specific three-dimensional spatial and electronic distribution. Any macromolecule having such specificity can be considered a receptor, whether the macromolecule is an enzyme, a protein, a glycoprotein, an antibody, an oligonucleotide sequence of DNA, RNA or the like. The various molecules that receptors bind are known as ligands.
Pharmaceutical drug discovery is one type of research that relies on the study of structure-function relationships. Most contemporary drug discovery involves discovering novel ligands with desirable patterns of specificity for biologically important receptors. Thus, the time necessary to bring new drugs to market could be greatly reduced by the discovery of novel methods which allow rapid screening of large numbers of potential ligands.
Since the introduction of solid phase synthesis methods for peptides and polynucleotides new methods employing solid phase strategies have been developed that are capable of generating thousands, and in some cases even millions, of individual peptide or nucleic acid polymers using automated or manual techniques. These synthesis strategies, which generate families or libraries of compounds, are generally referred to as “combinatorial chemistry” or “combinatorial synthesis” strategies.
Combinatorial chemistry strategies can be a powerful tool for rapidly elucidating novel ligands to receptors of interest. These methods show particular promise for identifying new therapeutics. See generally, Gorgon et al., “Applications of Combinatorial Technologies to Drug Discovery: II. Combinatorial Organic Synthesis, Library Screening Strategies, and Future Directions,” J. Med. Chem 37:1385-401 (1994) and Gallop et al., “Applications of Combinatorial Technologies to Drug Discovery: I. Background and Peptide Combinatorial Libraries,” J. Med. Chem 37:1233-51 (1994). For example, combinatorial libraries have been used to identify nucleic acid aptamers, Latham et al., “The Application of a Modified Nucleotide in Aptamer Selection: Novel Thrombin Aptamers Containing 5-(1-Pentynyl)-2′-Deoxy Uridine,” Nucl. Acids Res. 22:2817-2822 (1994); to identify RNA ligands to reverse transcriptase, Chen & Gold, “Selection of High-Affinity RNA Ligands to Reverse Transcriptase: Inhibition of CDNA Synthesis and RNase H Activity,” Biochemistry 33:8746-56 (1994); and to identify catalytic antibodies specific to a particular reaction transition state, Posner et al., “Catalytic Antibodies: Perusing Combinatorial Libraries,” Trends. Biochem. Sci. 19:145-50 (1994).
The diversity of libraries generated using combinatorial strategies is impressive. For example, these methods have been used to generate a library containing four trillion decapeptides, Pinilla et al., “Investigation of Antigen-Antibody Interactions Using a Soluble, Non-Support-Bound Synthetic Decapeptide Library Composed of Four Trillion (4×1012) Sequences,” Biochem. J. 301:847-53 (1994); 1,4-benzodiazepines libraries, Bunin et al., “The Combinatorial Synthesis and Chemical and Biological Evaluation of a 1,4-Benzodiazepine Library,” Proc. Natl. Acad. Sci. 91:4708-12 (1994) and U.S. Pat. No. 5,288,514, entitled “Solid Phase and Combinatorial Synthesis of Benzodiazepine Compounds on a Solid Support,” issued Feb. 22, 1994; libraries containing multiple small ligands tied together in the same molecules, Wallace et al., “A Multimeric Synthetic Peptide Combinatorial Library,” Pent. Res. 7:27-31 (1994); libraries of small organics, Chen et al., “‘Analogous’ Organic Synthesis of Compound Libraries: Validation of Combinatorial Chemistry in Small-Molecule Synthesis,” J. Am. Chem. Soc. 116:2661-2662 (1994); libraries of peptidosteroidal receptors, Boyce & Nestler, “Peptidosteroidal Receptors for Opioid Peptides: Sequence-Selective Binding Using a Synthetic Receptor Library,” J. Am. Chem. Soc. 116:7955-7956 (1994); and peptide libraries containing non-natural amino acids, Kerr et al;, “Encoded Combinatorial Peptide Libraries Containing Non-Natural Amino Acids,” J. Am. Chem. Soc. 115:2529-31 (1993).
To date, three general strategies for generating combinatorial libraries have emerged: “spatially-addressable,” “split-bead” and recombinant strategies.
These methods differ in one or more of the following aspects: reaction vessel design, polymer type and composition, control of physical constants such as time, temperature and atmosphere, isolation of products, solid-phase or solution-phase methods of assay, simple or complex mixtures, and method for elucidating the structure of the individual library members.
Of these general strategies, several sub-strategies have been developed. One spatially-addressable strategy that has emerged involves the generation of peptide libraries on immobilized pins that fit the dimensions of standard microtitre plates. See PCT Publication Nos. 91/17271 and 91/19818, each of which is incorporated herein by reference. This method has been used to identify peptides which mimic discontinuous epitopes, Geysen et al., BioMed. Chem. Lett. 3:391-404 (1993), and to generate benzodiazepine libraries, U.S. Pat. No. 5,288,514, entitled “Solid Phase and Combinatorial Synthesis of Benzodiazepine Compounds on a Solid Support,” issued Feb. 22, 1994 and Bunin et al., “The Combinatorial Synthesis and Chemical and Biological Evaluation of a 1,4-Benzodiazepine Library,” Proc. Natl. Acad. Sci. 91:4708-12 (1994). The structures of the individual library members can be decoded by analyzing the pin location in conjunction with the sequence of reaction steps used during the synthesis.
A second, related spatially-addressable strategy that has emerged involves solid-phase synthesis of polymers in individual reaction vesels, where the individual vessels are arranged into a single reaction unit. An illustrative example of such a reaction unit is a standard 96-well microtitre plate; the entire plate comprises the reaction unit and each well corresponds to a single reaction vessel. This approach is an extrapolation of traditional single-column solid-phase synthesis.
As is exemplified by the 96-well plate reaction unit, each reaction vessel is spatially defined by a two-dimensional matrix. Thus, the structures of individual library members can be decoded by analyzing the sequence of reactions to which each well was subjected.
Another spatially-addressable strategy employs “tea bags” to hold the synthesis resin. The reaction sequence to which each tea bag is subject is recorded, which determines the structure of the oligomer synthesized in each tea bag. See for example, Lam et al., “A New Type of Synthetic Peptide Library for Identifying Ligand-Binding Activity,” Nature 354:82-84 (1991); Houghten et al., “Generation and Use of Synthetic Peptide Combinatorial Libraries for Basic Research and Drug Discovery,” Nature 354:84-86 (1991); Houghten, “General Method for the Rapid Solid-Phase Synthesis of Large Numbers of Peptides: Specificity of Antigen-Antibody Interaction at the Level of Individual Amino Acids,” Proc. Natl. Acad. Sci. 82:5131-5135 (1985); and Jung et al., Agnew. Chem. Int. Ed. Engl. 91:367-383 (1992), each of which is incorporated herein by reference.
In another recent development, scientists combined the techniques of photolithography, chemistry and biology to create large collections of oligomers and other compounds on the surface of a substrate (this method is called “VLSIPS™”). See, for example, U.S. Pat. No. 5,143,854; PCT Publication No. 90/15070; PCT Publication No. 92/10092 entitled “Very Large Scale Immobilized Polymer Synthesis,” Jun. 25, 1992; Fodor et al., “Light-Directed Spatially Addressable Parallel Chemical Synthesis,” Science 251:767-773 (1991); Pease et al., “Light-Directed Oligonucleotide Arrays for Rapid DNA Sequence Analysis,” Proc. Natl. Acad. Sci. 91:5022-5026 (1994); and Jacobs & Fodor, “Combinatorial Chemistry: Applications of Light-Directed Chemical Synthesis,” Trends. Biotechnology 12(1): 19-26 (1994), each of which is incorporated herein by reference.
Others have developed recombinant methods for preparing collections of oligomers. See, for example, PCT Publication No. 91/17271; PCT Publication No. 91/19818; Scott, “Discovering Peptide Ligands Using Epitope Libraries,” TIBS 17:241-245 (1992); Cwirla et al., “Peptides on Phage: A Vast Library of Peptides for Identifying Ligands,” Proc. Natl. Acad. Sci. 87:6378-6382 (1990); Devlin et al., “Random Peptide Libraries: A Source of Specific Protein Binding Molecules,” Science 249:404-406 (1990); and Scott & Smith, “Searching for Peptide Ligands with an Epitope Library,” Science 249:386-390 (1990). Using these methods, one can identify each oligomer in the library by determining the coding sequences in the recombinant organism or phage. However, since the library members are generated in vivo, recombinant methods are limited to polymers whose synthesis is mediated in the cell. Thus, these methods typically have been restricted to constructing peptide libraries.
A third general strategy that has emerged involves the use of “split-bead” combinatorial synthesis strategies. See, for example, Furka et al., Int. J. Pept. Protein Res. 37:487-493 (1991), which is incorporated herein by reference. In this method synthesis supports are apportioned into aliquots, each aliquot exposed to a monomer, and the beads pooled. The beads are then mixed, reapportioned into aliquots, and exposed to a second monomer. The process is repeated until the desired library is generated.
Since the polymer libraries generated with the split-bead method are not spatially-addressable, the structures of the individual library members cannot be elucidated by analyzing the reaction histogram. Rather, structures must be determined by analyzing the polymers directly. Thus, one limitation of the split-bead approach is the requisite for an available means to analyze the polymer composition. While sequencing techniques are available for peptides and nucleic acids, sequencing reactions for polymers of other composition, such as for example carbohydrates, organics, peptide nucleic acids or mixed polymers may not be readily known.
Variations on the “split-bead” scheme have emerged that obviate the need to sequence the library member directly. These methods utilize chemicals to tag the growing polymers with a unique identification tag (“co-synthesis” strategies). See, for example, PCT Publication No. WO 94/08051 entitled “Complex Combinatorial Chemical Libraries Encoded with Tags,” Apr. 14, 1994; Nestler et al., “A General Method for Molecular Tagging of Encoded Combinatorial Chemistry Libraries,” J. Org. Chem. 59:4723-4724 (1994); PCT Publication No. WO 93/06121 entitled “Method of Synthesizing Diverse Collections of Oligomers,” Apr. 1, 1993; Needels et al., Proc. Natl. Acad. Sci. 90:10700-10704 (1993); Kerr et al., “Encoded Combinatorial Peptide Libraries Containing Non-Natural Amino Acids,” J. Amer. Chem. Soc. 115:2529-2531 (1993); and Brenner & Lerner, “Encoded Combinatorial Chemistry,” Proc. Natl. Acad. Sci. 89:5381-5383 (1992), each of which is incorporated herein by reference.
Encoding library members with chemical tags occurs in such a fashion that unique identifiers of the chemical structures of the individual library members are constructed in parallel, or are co-synthesized, with the library members. Typically, in a linear three component synthesis containing building blocks A, B and C in the process of generating library member ABC, an encoding. tag is introduced, at each stage such that the tags TA, TB and TC would encode for individual inputs in the library. The synthesis would proceed as follows: (a) Chemical A is coupled onto a synthesis bead, immediately followed by coupling tag TA to the bead; (b) The bead is subject to deprotection conditions which remove the protecting group selectively from A, leaving TA protected. Chemical B is coupled to the bead, generating the sequence AB. The bead is then subject to deprotection which selectively removes the protecting group from TA, and TB is coupled to the bead, generating tag sequence TATB; (c) The third component C and concomitant tag TC is added to the bead in the manner described above, generating library sequence ABC and tag sequence TATBTC.
For large libraries containing three chemical inputs, the chemical tagging sequence is the same. Thus, to generate a large library containing the complete set of three-input, one hundred unit length polymers, or 1003=106 library members, unique identifying tags are introduced such that there is a unique identifier tag for each different chemical structure. Theoretically, this method is applicable to libraries of any complexity as long as tagging sequences can be developed that have at least the same number of identification tags as there are numbers of unique chemical structures in the library.
While combinatorial synthesis strategies provide a powerful means for rapidly identifying target molecules, substantial problems remain. For example, since members of spatially addressable libraries must be synthesized in spatially segregated arrays, only relatively small libraries can be constructed. The position of each reaction vessel in a spatially addressable library is defined by an XY coordinate pair such that the entire library is defined by a two-dimensional matrix. As the size of the library increases the dimensions of the two-dimensional matrix increases. In addition, as the number of different transformation events used to construct the library increases linearly, the library size increases exponentially. Thus, while generating the complete set of linear tetramers comprised of four different inputs requires only a 16×16 matrix (44=256 library members), generating the complete set of linear octamers composed of four different inputs requires a 256×256 matrix (48=65,536 library members), and generating the complete set of linear tetramers composed of twenty different inputs requires a 400×400 matrix (204=160,000 library members). Therefore, not only does the physical size of the library matrix quickly become unwieldy (constructing the complete set of linear tetramers composed of twenty different inputs using spatially-addressable techniques requires 1667 microtitre plates), delivering reagents to each reaction vessel in the matrix requires either tedious, time-consuming manual manipulations, or complex, expensive automated equipment.
While the VLSIPS™ method attempts to overcome this limitation through miniaturization, VLSIPS™ requires specialized photoblocking chemistry, expensive, specialized synthesis equipment and expensive, specialized assay equipment. Thus, the VLSIPS™ method is not readily and economically adaptable to emerging solid phase chemistries and assay methodologies.
Split bead methods also suffer severe limitations. Although large libraries can theoretically be constructed using split-bead methods, the identity of library members displaying a desirable property must be determined by analytical chemistry. Accordingly, split-bead methods can only be employed to synthesize compounds that can be readily elucidated by microscale sequencing, such as polypeptides and polynucleotides.
Co-synthesis strategies have attempted to solve this structure elucidation problem. However, these methods also suffer limitations. For example, the tagging structures may be incompatible with synthetic organic chemistry reagents and conditions. Additional limitations follow from the necessity for compatible protecting groups which allow the alternating co-synthesis of tag and library member, and assay confusion that may arise from the tags selectively binding to the assay receptor.
Finally, since methods such as the preceding typically require the addition of like moieties, there is substantial interest in discovering methods for producing labeled libraries of compounds which are not limited to sequential addition of like moieties, and which are amenable to any chemistries now known or that will be later developed to generate chemical libraries. Such methods would find application, for example, in the modification of steroids, sugars, co-enzymes, enzyme inhibitors, ligands and the like, which frequently involve a multi-stage synthesis in which one would wish to vary the reagents and/or conditions to provide a variety of compounds.
In such methods the reagents may be organic or inorganic reagents, where functionalities or side groups may be introduced, removed or modified, rings opened or closed, stereochemistry changed, and the like.
From the above, one can recognize that there is substantial interest in developing improved methods and apparatus for the synthesis of complex labeled combinatorial chemical libraries which readily permit the construction of libraries of virtually any composition and which readily permit accurate structural determination of individual compounds within the library that are identified as being of interest. Many of the disadvantages of the previously-described methods as well as many of the needs not met by them are addressed by the present invention, which as described more fully hereinafter, provides myriad advantages over these previously-described methods.
Glossary
The following terms are intended to have the following general meanings as they are used herein:
Labeled Synthetic Oligomer Library: A “labeled synthetic oligomer library” is a collection of random synthetic oligomers wherein each member of such a library is labeled with a unique identifier tag from which the structure or sequence of each oligomer can be deduced.
Identifier Tag: An “identifier tag” is any detectable attribute that provides a means whereby one can elucidate the structure of an individual oligomer in a labeled synthetic oligomer library. Thus, an identifier tag identifies which transformation events an individual oligomer has experienced in the synthesis of a labeled synthetic oligomer library, and at which reaction cycle in a series of synthesis cycles each transformation event was experienced.
An identifier tag may be any detectable feature, including, for example: a differential absorbance or emission of light; magnetic or electronically pre-encoded information; or any other distinctive mark with the required information. An identifier tag may be pre-encoded with unique identifier information prior to synthesis of a labeled synthetic oligomer library, or may be encoded with a identifier information in concomitant with synthesis of a labeled synthetic oligomer library.
In this latter embodiment, the identifier information added at each synthesis cycle is preferably added in a sequential fashion, such as, for example digital information, with the identifier information identifying the transformation event of synthesis cycle two being appended onto the identifier information identifying the transformation event of synthesis cycle one, and so forth.
Preferably, an identifier tag is impervious to the reaction conditions used to construct the labeled synthetic oligomer library.
A preferred example of an identifier tag is a microchip that is pre-encoded or encodable with information, which information is related back to a detector when the microchip is pulsed with electromagnetic radiation.
Pre-Encoded Identifier Tag: A “pre-encoded identifier tag” is an identifier tag that is pre-encoded with unique identifier information prior to synthesis of a labeled synthetic oligomer library. A preferred example of such a pre-encoded identifier tag is a microchip that is pre-encoded with information, which information is related back to a detector when the microchip is pulsed with electromagnetic radiation.
Encodable Identifier Tag: An “encodable identifier tag” is an identifier tag that is capable of receiving identifier information from time to time. An encodable identifier tag may or may not be pre-encoded with partial or complete identifier information prior to synthesis of a labeled synthetic oligomer library. A preferred example of such an encodable identifier tag is a microchip that is capable of receiving and storing information from time to time, which information is related back to a detector when the microchip is pulsed with electromagnetic radiation.
Transformation Event: As used herein, a “transformation event” is any event that results in a change of chemical structure of an oligomer or polymer. A “transformation event” may be mediated by physical, chemical, enzymatic, biologicall or other means, or a combination of means, including but not limited to, photo, chemical, enzymatic or biologically mediated isomerization or cleavage; photo, chemical, enzymatic or biologically mediated side group or functional group addition, removal or modification; changes in temperature; changes in pressure; and the like. Thus, “transformation event” includes, but is not limited to, events that result in an increase in molecular weight of an oligomer or polymer, such as, for example, addition of one or a plurality of monomers, addition of solvent or gas, or coordination of metal or other inorganic substrates such as, for example, zeolites; events that result in a decrease in molecular weight of an oligomer or polymer, such as, for example, de-hydrogenation of an alcohol to from an alkene or enzymatic hydrolysis of an ester or amide; events that result in no net change in molecular weight of an oligomer or polymer, such as, for example, stereochemistry changes at one or a plurality of a chiral centers, Claissen rearrangement, or Cope rearrangement; and other events as will become apparent to those skilled in the art upon review of this disclosure. See, for example, application Ser. No. 08/180,863 filed Jan. 13, 1994, which is assigned to the assignee of the present invention and PCT Publication WO 94/08051 entitled “Complex Combinatorial Libraries Encoded with Tags,” Apr. 14 (1994), each of which is incorporated herein by reference.
Monomer: As used herein, a “monomer” is any atom or molecule capable of forming at least one chemical bond. Thus, a “monomer” is any member of the set of atoms or molecules that can be joined together as single units in a multiple of sequential or concerted chemical or enzymatic reaction steps to form an oligomer or polymer. Monomers may have one or a plurality of functional groups, which functional groups may be, but need not be, identical.
The set of monomers useful in the present invention includes, but is not restricted to, alkyl and aryl amines; alkyl and aryl mercaptans; alkyl and aryl ketones; alkyl and aryl carboxylic acids; alkyl and aryl ester; alkyl and aryl ethers; alkyl and aryl sulfoxides; alkyl and aryl sulfones; alkyl and aryl sulfonamides; phenols; alkyl alcohols; alkyl and aryl alkenes; alkyl and aryl lactams; alkyl and aryl lactones; alkyl and aryl di- and polyenes; alkyl and aryl alkynes; alkyl and aryl unsaturated ketones; aldehydes; sulfoxides; sulfones; heteroatomic compounds containing one or more of the atoms of: nitrogen, sulfur, phosphorous, oxygen, and other polyfunctional molecules containing one or more of the above functional groups; L-amino acids; D-amino acids; deoxyribonucleosides; deoxyribonucleotides; ribonucleosides; ribonucleotides; sugars; benzodiazenines; β-lactams; hydantoins; quinones; hydroquinones; terpenes; and the like.
The monomers of the present invention may have groups protecting the functional groups within the monomer. Suitable protecting groups will depend on the functionality and particular chemistry used to construct the library. Examples of suitable functional protecting groups will be readily apparent to skilled artisans, and are described, for example, in Greene and Wutz, Protecting Groups in Organic Synthesis, 2d ed., John Wiley & Sons, NY (1991), which is incorporated herein by reference.
As used herein, “monomer” refers to any member of a basis set for synthesis of an oligomer. For example, dimers of L-amino acids form a basis set of 400 “monomers” for synthesis of polypeptides. Different basis sets of monomers may be used at successive steps in the synthesis of a polymer.
Oligomer or Polymer: As used herein, an “oligomer” or “polymer” is any chemical structure that can be synthesized using the combinatorial library methods of this invention, including, for example, amides, esters, thioethers, ketones, ethers, sulfoxides, sulfonamides, sulfones, phosphates, alcohols, aldehydes, alkenes, alkynes, aromatics, polyaromatics, heterocyclic compounds containing one or more of the atoms of: nitrogen, sulfur, oxygen, and phosphorous, and the like; chemical entities having a common core structure such as, for example, terpenes, steroids, β-lactams, benzodiazepines, xanthates, indoles, indolones, lactones, lactams, hydantoins, quinones, hydroquinones, and the like; chains of repeating monomer units such as polysaccharides, phospholipids, polyurethanes, polyesters, polycarbonates, poly ureas, polyamides, polyethyleneimines, poly arylene sulfides, polyimides, polyacetates, polypeptides, polynucleotides, and the like; or other oligomers or polymers as will be readily apparent to one skilled in the art upon review of this disclosure. Thus, an “oligomer” and “polymer” of the present invention may be linear, branched, cyclic, or assume various other forms as will be apparent to those skilled in the art.
Concerted: As used herein “concerted” means synchronous and asynchronous formation of one or more chemical bonds in a single reaction step.
Substrate: As used herein, a “substrate” is a synthesis means linked to an identifier tag. By way of example and not limitation, a “substrate” may be an identifier tag functionalized with one or a plurality of groups or linkers suitable for synthesis; a glass or polymer encased identifier tag, which glass or polymer is functionalized with one or a plurality of groups or linkers suitable for synthesis; an identifier tag that is coated with one or a plurality of synthesis supports; an identifier tag retained within a frame or housing, which frame or housing is functionalized with one or a plurality of groups or linkers suitable for synthesis; an identifier tag retained within a frame or housing, which frame or housing also retains one or a plurality of synthesis supports; and the like.
Synthesis Means: A “synthetic means” is any means for carrying out synthesis of a labeled synthetic oligomer library. Thus, “synthesis means” may comprise reaction vessels, columns, capillaries, frames, housings, and the like, suitable for carrying out synthesis reactions; one or a plurality of synthesis supports suitable for carrying out synthesis reactions; or functional groups or linkers attached to an identifier tag suitable for carrying out synthesis reactions.
“Synthesis means” may be constructed such they are capable of retaining identifier tags and/or synthesis supports.
In a preferred embodiment a “synthesis means” is one or a plurality of synthesis supports.
Synthesis Support: A “synthesis support” is a material having a rigid or semi-rigid surface and having functional groups or linkers, or that is capable of being derivatized with functional groups or linkers, that are suitable for carrying out synthesis reactions.
Such materials will preferably take the form of small beads, pellets, disks, capillaries, hollow fibers, needles, solid fibers, cellulose beads, pore-glass beads, silica gels, polystyrene beads optionally cross-linked with polyethylene glycol divinylbenzene, grafted co-poly beads, polyacrylamide beads, latex beads, dimethylacrylamide beads optionally cross-linked with N,N′-bis-acryloyl ethylene diamine, glass particles coated with a hydrophobic polymer, or other convenient forms.
“Synthesis supports” may be constructed such that they are capable of retaining identifier tags.
Linker: A “linker” is a moiety, molecule, or group of molecules attached to a synthesis support or substrate and spacing a synthesized polymer or oligomer from the synthesis support or substrate. A “linker” can also be a moiety, molecule, or group of molecules attached to a substrate and spacing a synthesis support from the substrate.
Typically a linker will be bi-functional, wherein said linker has a functional group at one end capable of attaching to a monomer, oligomer, synthesis support or substrate, a series of spacer residues, and a functional group at another end capable of attaching to a monomer, oligomer, synthesis support or substrate. The functional groups may be, but need not be, identical.
Spacer residues: “Spacer residues” are atoms or molecules positioned between the functional groups of a bifunctional linker, or between a functional group of a linker and the moiety to which the linker is attached. “Spacer residues” may be atoms capable of forming at least two covalent bonds such as carbon, silicon, oxygen, sulfur, phosphorous, and the like, or may be molecules capable of forming at least two covalent bonds such as amino acids, peptides, nucleosides, nucleotides, sugars, carbohydrates, aromatic rings, hydrocarbon rings, linear and branched hydrocarbons, and the like.
Linked together the spacer residues may be rigid, semi-rigid or flexible. Linked spacer residues may be, but need not be, identical.
Pre-encoded Substrate: A “pre-encoded substrate” is a substrate wherein the identifier tag is a pre-encoded identifier tag.
Encodable Substrate: An “encodable substrate” is a substrate wherein the identifier tag is an encodable identifier tag.
Synthetic: A compound is “synthetic” when produced by in vitro chemical or enzymatic synthesis.
Oligomer or Polymer Sequence: As used herein “oligomer sequence” or “polymer sequence” refers to the chemical structure of an oligomer or polymer.